Systems and methods for cellular separation

ABSTRACT

Methods and systems disclosed herein provide systems and methods for maintaining sterile conditions inside of a biological-fluid container during an entire process of delivery, incubation, centrifugation, and extraction of fluid. The methods and systems also provide systems and methods for localized extraction of a portion of a biological fluid.

BACKGROUND

The disclosure herein generally relates to cellular-separation systemsand, more particularly, to caps for biological-fluid containers ofcellular-separation systems.

SUMMARY

Typical cellular-separation systems allow for insertion of biologicalfluid into a biological-fluid container and extraction of a portion ofthe biological fluid from the biological-fluid container. However,current cellular-separation systems have various drawbacks. Forinstance, existing systems do not always allow for maintaining sterileconditions inside of the biological-fluid container during the entireprocess of delivery, incubation, centrifugation, and extraction of fluidfrom the biological-fluid container. Further, it is difficult and/or notpossible in existing systems to reach particular points within thebiological-fluid container for localized extraction of a portion of abiological fluid.

Methods and systems in accordance with the present disclosure providesystems and methods for maintaining sterile conditions inside of abiological-fluid container during an entire process of delivery,incubation, centrifugation, and extraction of fluid. Methods and systemsin accordance with the present disclosure also provide systems andmethods for localized extraction of a portion of a biological fluid.

In an example, a cap for a biological-fluid container is described. Acap includes an injection port configured for injection of biologicalfluid through the injection port and an extraction port configured forextraction of biological fluid through the extraction port. A cap canalso include an injection-port cap configured to attach to the injectionport and an extraction-port cap configured to attach to the extractionport, wherein at least one of the injection-port cap or theextraction-port cap is a vented cap. Further, a cap can include apenetrable flexible seal configured for insertion and extraction ofbiological fluid through the flexible seal.

In another example, a cellular-separation system is described. Acellular-separation system includes a biological-fluid container and acap attached to the biological-fluid container. A cap can include aninjection port configured for injection of biological fluid through theinjection port and an extraction port configured for extraction ofbiological fluid through the extraction port. A cap can also include aninjection-port cap configured to attach to the injection port and anextraction-port cap configured to attach to the extraction port, whereinat least one of the injection-port cap or the extraction-port cap is avented cap. Further, a cap can include a penetrable flexible sealconfigured for insertion and extraction of biological fluid through theflexible seal.

In another example, a method to isolate one or more fractions of abiological fluid is described. A method includes injecting a biologicalfluid into a biological-fluid container through an injection port in acap of a cellular-separation system in accordance with the presentdisclosure, wherein the injection port is configured to attach to avented cap. A method can also include incubating a biological fluidinjected into a biological-fluid container and centrifuging thebiological-fluid container to form two or more fractions of thebiological fluid. Further, a method can include removing (e.g., via aneedle) one or more fractions of the biological fluid from thebiological-fluid container through the penetrable flexible seal in thecap, wherein during said removing the vented cap is attached to theinjection port and releases pressure from the biological-fluidcontainer.

The features, functions, and advantages that have been discussed can beachieved independently in various embodiments or can be combined in yetother embodiments further details of which can be seen with reference tothe following description and drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a illustrates a perspective view of an example cellular-separationsystem, according to an example embodiment.

FIG. 1b illustrates an exploded, side view of the examplecellular-separation system of FIG. 1a , according to an exampleembodiment.

FIG. 1c illustrates another exploded, side view of the examplecellular-separation system of FIG. 1a when the vented caps are attachedto the injection and extraction ports, according to an exampleembodiment.

FIG. 2 illustrates an example syringe that can be used for injectionand/or extraction of a biological fluid from the examplecellular-separation system of FIG. 1a , according to an exampleembodiment.

FIG. 3a illustrates a cross-sectional, side view of the examplecellular-separation system of FIG. 1a after insertion of a biologicalfluid, according to an example embodiment.

FIG. 3b illustrates a cross-sectional, side view of the examplecellular-separation system of FIG. 1a after incubation andcentrifugation, according to an example embodiment.

FIG. 4 illustrates an example needle that can be used for injectionand/or extraction of a biological fluid from the examplecellular-separation system of FIG. 1a , according to an exampleembodiment.

FIG. 5 illustrates a top view of the cap of the examplecellular-separation system of FIG. 1a , according to an exampleembodiment.

FIG. 6 illustrates an example non-vented cap that can be used in theexample cellular-separation system of FIG. 1a , according to an exampleembodiment.

FIG. 7 illustrates an example centrifuge that can be used to centrifugethe cellular-separation system of FIG. 1a , according to an exampleembodiment.

DETAILED DESCRIPTION

Disclosed embodiments will now be described more fully hereinafter withreference to the accompanying drawings, in which some, but not all ofthe disclosed embodiments are shown. Indeed, several differentembodiments may be described and should not be construed as limited tothe embodiments set forth herein.

As mentioned above, current cellular-separation systems have variousdrawbacks. Methods and systems in accordance with the present disclosureprovide methods and systems for allowing for maintenance of sterileconditions inside of a biological-fluid container during the entireprocess of delivery, incubation, centrifugation, and extraction ofbiological fluid(s) from the biological-fluid container. Furthermore,methods and systems in accordance with the present disclosure alsoprovide systems and methods for localized extraction of a portion of abiological fluid from the biological-fluid container.

Referring now to FIGS. 1a-b , an example cellular-separation system 100is illustrated. The cellular-separation system 100 includes abiological-fluid container 102 and a cap 104 attached to thebiological-fluid container 102. The cap includes an injection port 106to allow for injection of biological fluid through the injection portand an extraction port 108 to allow for extraction of biological fluidthrough the extraction port. The cap 104 also includes an injection-portcap configured to attach to the injection port 106 and anextraction-port cap configured to attach to the extraction port 108,wherein at least one of the injection-port cap or the extraction-portcap is a vented cap. In this example of FIGS. 1a-b , the injection-portcap is first vented cap 110 and the extraction-port cap is second ventedcap 112. Further, the cap 104 includes a penetrable flexible seal 114 toallow for insertion and extraction of biological fluid through thepenetrable flexible seal.

Any suitable materials can be used for these components of thecellular-separation system 100 including but not limited to plastic,rubber, and/or metal. In an example embodiment, the biological-fluidcontainer 102, injection port 106, extraction port 108, first vented cap110, and second vented cap 112 comprise a medical-grade plastic, such aspolypropylene. Other materials are possible as well. Further, in anexample embodiment, the penetrable flexible seal 114 comprises a rubbersuch as bromobutyl or polyisoprene. As a general matter, the penetrableflexible seal 114 comprises a seal that can be penetrated by aninstrument (e.g., a needle) and can maintain a hermetic seal when thatinstrument is removed. In an example embodiment, the penetrable flexibleseal 114 is a rubber stopper or gasket. Other example penetrableflexible seals and materials are possible as well.

The cap 104 includes a base 116 configured to attach to thebiological-fluid container 102. In an example embodiment, the base 116includes threads to attach to corresponding threads on thebiological-fluid container 102. Additionally or alternatively, the base116 is attached to the biological-fluid container 102 with an adhesive.In an example embodiment, the base 116 is welded to the biological-fluidcontainer 102. Other examples are possible as well. The cap 104 providesa hermetic seal for the biological-fluid container 102 when the base 116is attached to the biological-fluid container.

The injection port 106 includes a connection fitting 122 configured toattach directly to a syringe, such as syringe 124 shown in FIG. 2. Theconnection fitting 122 is also configured to attach to the first ventedcap 110 as shown in FIG. 1c . Similarly, the extraction port 108includes a connection fitting 126 configured to attach directly to asyringe such as syringe 124. The connection fitting 126 is alsoconfigured to attach to the second vented cap 112 as shown in FIG. 1 c.

In an example embodiment, the syringe 124 includes a male luer lock 130and the vented caps 110, 112 are vented male luer caps, whereasconnection fittings 122, 126 are female luer locks. In other examples,however, the connection fittings 122, 126 are male luer locks, whereasthe syringe 124 includes a female luer lock and the vented caps 110, 112are vented female luer locks. Other examples are possible as well.

In an example embodiment, the connection fitting 122 includes threading128 (see FIG. 1a ) to accommodate the first vented cap 110 and syringe124, and connection fitting 126 includes threading 129 (see FIG. 1a ) toaccommodate second vented cap 112 and syringe 124. This threading allowsthe syringe 124 or the vented caps 110, 112 to be twisted on theinjection port 106 and extraction port 108 during the connectionprocess.

In the example of FIGS. 1a-c , the first vented cap 110 is tethered tothe injection port—106 and the second vented cap 112 is tethered to theextraction port 108. This tethering helps to ensure that the vented caps110, 112 remained attached to the cap 104 and are not misplaced when thevented caps 110, 112 are detached from the ports 106, 108 (e.g., inorder to attach syringe 124 to ports 106, 108). However, in otherexamples, the vented caps 110, 112 can be standalone caps that are nottethered to the ports 106, 108.

In operation, the cellular-separation system 100 can be used forseparating components of biological fluid (e.g., separating autologouscells or tissues from blood). In an example embodiment, thecellular-separation system 100 includes an element(s) designed toisolate or concentrate the desired component(s). For instance, in theexample shown in FIGS. 1a-c , the cellular-separation system 100includes a plurality of beads 132 in the biological-fluid container 102to activate Interleukin-1 Receptor Antagonist Protein (IL-1Ra)production in blood. Other examples are possible as well. Further,although three beads 132 are illustrated, more or fewer beads arepossible as well (e.g., about 5, 10, 20, 30, 40, 50, or higher).

In an example embodiment, the beads 132 are manufactured from a glasslike composition such as, borosilicate glass, alumina, silicate, quartz,bioglass, ceramic glass, flint glass, fluorosilicate glass,phosphosilicate glass, and cobalt glass or conundrum. In an exampleembodiment, the beads have a spherical shape to provide for a maximumsurface area for blood contact. In an alternate embodiment, thebiological-fluid container 102 can contain gels, wool, powder, plastic,granules or fibers. The beads can be provided with a coating to maximizethe production of IL-1Ra by monocytes within blood. The coating can besilane, surfactants, polyether, polyester, polyurethane, or polyolgroups. The beads can range in size from 0.1-5 mm; however, other sizesare possible as well. In a particular example, the beads 132 are about3.0 mm. Optimal production of IL-1Ra occurs when the maximum surfacearea of the beads is exposed to the blood within the biological-fluidcontainer 102. A maximum amount of blood in the biological-fluidcontainer 102 helps to optimize the production of IL-1Ra. In order toaccomplish both goals, the volume of the beads should be minimized toaccomplish the maximum exposed surface area. Accordingly, the diameterof the beads has been tailored to maximize the volume of injected bloodor biological fluid in the container and maximize the surface area forblood/bead contact.

After a biological fluid is injected into the biological-fluid container102 optionally with beads 132, the biological fluid can be incubated foran appropriate period of time. In an example embodiment, the biologicalfluid is incubated for a sufficient time and at a sufficient temperatureto produce a therapeutically active protein in the biological fluid.After incubation, the biological fluid can be placed into a centrifuge(e.g., such as centrifuge 182 shown in FIG. 7) to separate components ofthe biological fluid into two or more fractions. For instance, FIG. 3aillustrates cellular-separation system 100 with biological fluid 140 inthe biological-fluid container 102 prior to incubation andcentrifugation, and FIG. 3b illustrates cellular-separation system 100after incubation and centrifugation. The biological fluid 140 can be abiological fluid from a mammal, e.g., blood. In one example, as seen bycomparing these Figures, after incubation and centrifugation, thebiological fluid (in this case blood 140) can be separated into a serumfraction 142 and a fraction of the remainder 144 of the blood 140. Asthe beads 132 (see FIG. 1b ) within the biological-fluid container canbe designed to activate IL-1Ra production in blood, the serum fraction142 can have increased anti-inflammatory and regenerative proteinconcentration levels. The serum fraction 142 can then be extracted fromthe biological-fluid container and can be used for treatment (e.g.,treatment of a tissue injury in the mammal from which blood 140 wasextracted).

Beneficially, the cap 104 provides numerous ways to both (i) injectbiological fluid into the biological-fluid container 102 and (ii)extract the biological fluid (and particularly desired components ofthat biological fluid, such as serum 142) from the biological-fluidcontainer 102. More particularly, the cap 104 allows for injection andextraction of the biological fluid via a needle, as well as for a directsyringe attachment for injection and extraction of biological fluid. Byproviding these numerous ways to both inject and extract the biologicalfluid, both insertion and extraction of the biological fluid can betailored based on the particular application and/or cellular componentsbeing separated. In practice, some applications can be better suited forinsertion and/or extraction via a direct syringe attachment, whereasother applications can be better suited for insertion and/or extractionvia a needle. Further, by providing both the penetrable flexible seal114 and the vented caps 110, 112 in the cap 104, the cap 104 can allowfor maintenance of sterility inside of the biological-fluid containerthroughout the entire delivery, incubation, centrifugation, andextraction process. The sterility can be important where incubationperiods are long (e.g., about 6, 8, 12, 24, 36 or 48 hours) attemperatures conducive to microbial growth and multiplication.

The first and second vented caps 110, 112 are configured to releasepressure from the biological-fluid container 102 during at least one ofinsertion or extraction of biological fluid through the penetrableflexible seal 114 via a needle. The vented caps 110, 112 help to provideproper air pressure so as to allow for the insertion and extraction ofthe biological fluid (e.g., blood 140 or serum 142) through thepenetrable flexible seal 114 via a needle. The vented caps 110, 112 caninclude one-way release valves that are pressure activated. When thepressure reaches a threshold level, the release valves can open to allowventing. In an example embodiment, the threshold level is about 5 poundsper square inch (psi). However, in other examples, the threshold can behigher or lower than about 5 psi (e.g., about 3, 4, 5, 6, 7, 8 psi). Thevented caps 110, 112 can include a plurality of pores that are sized toallow air to flow through the pores but prevent the biological fluidfrom flowing through the pores. In an example embodiment, the size ofthe pores ranges between 0.2-10 μm (e.g., about 0.2, 0.45, 1.2, 3, 10μm). Other sizes are possible as well. Since the biological-fluidcontainer 102 is a pressurized container when hermetically sealed by cap104, it would be difficult to inject into or extract fluid from thecontainer without the venting provided by vented cap 110 and/or ventedcap 112.

In order to insert and/or extract biological fluid via a needle, such asneedle 134 (see FIG. 4), the needle 134 can puncture the penetrableflexible seal 114 and be inserted into the biological-fluid container102. Since both insertion and extraction of biological fluid through thepenetrable flexible seal 114 are possible, sterility can be maintainedinside of the biological-fluid container throughout the entire delivery,incubation, centrifugation, and extraction process. Additionally, in anexample embodiment, needle 134 can also be inserted through the ventedcaps 110, 112 for insertion and/or extraction of biological fluidthrough the vented caps 110, 112. In this example, vented caps 110, 112can include a penetrable filter that provides venting and also allowsfor insertion of needle 134. Any suitable materials can be used for thepenetrable filter including but not limited to acrylic copolymer,polytetrafluoroethylene (PTFE), nylon, polyurethane, and/orpolyethersulfone. Other materials are possible as well. These penetrablefilters of vented caps 110, 112 can also help to maintain sterilityinside of the biological-fluid container throughout the entire delivery,incubation, centrifugation, and extraction process.

As mentioned above, the cap 104 also allows for a direct syringeattachment for injection and extraction of biological fluid. Inparticular, vented caps 110, 112 can be detached from the injection port106 and extraction port 108, respectively (as shown in FIGS. 1a-b ), andsyringe 124 can then be directly attached to the ports 106, 108 forinsertion and extraction of biological fluid. In an example embodiment,direct syringe attachment can be suitable for large volume aspirations,when a needle is unavailable, and/or when a needle is unwanted due todisposal concerns.

In an example embodiment, the injection port 106 and extraction port 108include cannulas or conduits configured to extend into thebiological-fluid container 102. For instance, as shown in FIG. 1c ,injection port 106 includes cannula 152 extending into thebiological-fluid container 102, and extraction port 108 includes cannula154 extending into the biological-fluid container 102. These cannulas152, 154 help with the insertion and extraction of biological fluidusing syringe 124. A cannula 152 can include a beveled edge 156 that canhelp to direct the flow of biological fluid towards the sidewall 158 ofthe tubular body 160 of the container (rather than allowing the fluid toflow directly to the bottom 162. This beveled edge 156 can help toprevent hemolysis. Further, by extending into the biological-fluidcontainer 102, a cannula 154 can help with the extraction of biologicalfluid by a direct syringe attachment. For instance, a cannula 152 canextend into serum 142 (as is the case in FIG. 3b ), and this extensioninto serum 142 can help syringe 124 suction serum 142 from thebiological-fluid container 102 to the syringe 124. In an example,cannula 154 can also include a beveled edge.

The biological-fluid container 102 can include a tubular body. However,in other examples embodiments, other shapes of the body are possible aswell. In general, the biological-fluid container 102 has a size andshape that allows the biological-fluid container 102 to fit into acentrifuge. In an example, the biological-fluid container can 102 hold avolume of about 50 ml to about 60 ml although it could be any size thatwould fit within a centrifuge.

In addition to allowing for maintaining sterile conditions inside of abiological-fluid container throughout the entire delivery, incubation,centrifugation, and extraction process, the penetrable flexible seal 114also beneficially allows for a means of extraction that provides agreater range of maneuvering to facilitate extraction compared to meansof extraction in existing cellular-separation systems. For instance, theneedle 134 can be inserted to substantially any level within thebiological-fluid container 102 to extract any fraction or portion of thebiological fluid prior to or after centrifugation. Further, theorientation (e.g., angle) of the needle 134 can be adjusted so that theneedle 134 is able to reach all or substantially all of the pointswithin the biological-fluid container 102. For instance, the needle 134can be inserted to a given depth and can be swiveled about the axis ofthe penetrable flexible seal 114 so as to reach all or substantially allof the points within the biological-fluid container 102.

As a particular example, with reference to FIG. 3b , the needle 134 canbe inserted to the level of serum 142 for extraction of serum 142.Further, both the angle and depth of the needle 134 can be adjusted, soas to allow the needle to reach localized points within the serumfraction 142 or within any other portion or fraction of the biologicalfluid. For instance, the biological-fluid container 102 can includesidewalls 158, 166, and the serum fraction 142 (for example) can includea proximal point 168 and a distal point 170. The angle and depth of theneedle 134 can be adjusted to reach the proximal and distal points ofboth sidewalls 158, 166, as well as points there between. In an example,the needle 134 may be prevented from reaching points directly betweencannula 152 (if present) and sidewall 158 and between cannula 154 (ifpresent) and sidewall 166; however, all or most of the other points inthe biological-fluid container 102 can be reached.

Although in the example of FIG. 3b the biological fluid is illustratedas being separated into two fractions, in other examples the biologicalfluid can be separated into 3, 4, 5, 6, or more fractions. The needle134 can be inserted into the biological-fluid container 102 to anyfraction of any component to be extracted.

In an example, whole blood can be fractionated into an erythrocytelayer, a buffy layer, and a platelet poor plasma (PPP) layer viacentrifugation. A portion of the PPP fraction can be removed, and theremaining fluid can then be fractionated into other layers, such as anerythrocyte fraction and a plasma fraction (i.e., platelet rich plasma).In an embodiment, platelet rich plasma is removed from thebiological-fluid container and administered to a subject.

In an example, bone marrow can be fractionated into a platelet poorplasma layer, a buffy layer, and an erythrocyte/granulocyte layer withinthe biological-fluid container via centrifugation. Optionally, at leasta portion of the platelet poor plasma layer can be removed from thebiological-fluid container. The platelet poor plasma layer and the buffylayer can be centrifuged wherein the platelet poor plasma layer and thebuffy layer fractionates into a bone marrow mononuclear cell fractionand an erythrocyte/granulocyte fraction. The bone marrow mononuclearcell fraction can be removed from the biological-fluid container andadministered to a subject.

In an example, adipose tissue can be fractionated (top to bottom) into alipid layer, a compressed adipose layer, and an excess fluid layerwithin the biological-fluid container via centrifugation. At least aportion of the lipid layers and excess fluid layers can be removed frombiological-fluid container. The fluid can be centrifuged again such thatthe fluid fractionates into a top adipocyte layer and a bottom stromalvascular fraction. The stromal vascular fraction can be removed from thebiological-fluid container and used in the treatment of soft tissueinjury or damage. Other examples are possible as well.

As mentioned above, needle 134 can also be inserted through a firstvented cap 110 and/or a second vented cap 112. Although needle 134 canbe inserted through a vented cap 110, the range of motion of the needle134 can be limited by the structure of the vented cap 110 and/or cannula152 of the injection port 106. Similarly, although needle 134 can beinserted through the second vented cap 112, the range of motion of theneedle 134 can be limited by the structure of the second vented cap 112and/or cannula 154 of the extraction port 108. On the other hand, thepenetrable flexible seal 114 allows for a greater range of movement ofthe needle 134 within the biological-fluid container 102, thus allowingfor localized extraction of the biological fluid from all orsubstantially all of the points within the biological-fluid container102.

The penetrable flexible seal 114 can be positioned on the cap 104 in anysuitable location on the cap 104. In an example, the position of thepenetrable flexible seal 114 on the cap 104 is selected so as tomaximize the range of motion of the needle 134 when it is inserted intothe penetrable flexible seal 114. With reference to FIG. 5, whichillustrates a top view of the cap 104, the penetrable flexible seal 114is positioned in the center or substantially in the center of the cap104. This allows for a large range of motion for needle 134 when it isinserted into the penetrable flexible seal 114 to a desired depth andits orientation (e.g., angle) is adjusted for a desired localizedextraction. However, other positions of the penetrable flexible seal 114are possible as well. Furthermore, in other example embodiments, the cap104 includes a plurality of penetrable flexible seals (e.g., about 2, 3,4, 5, or more). The number and/or location of the plurality ofpenetrable flexible seals can be selected so as to maximize the reach ofthe needle 134 within the biological-fluid container 102.

Although in the example of FIGS. 1a-c , the cap 104 includes vented capson both the injection port 106 and extraction port 108, in otherexamples the cap 104 can include one vented cap for the injection port106 or the extraction port 108. In this example, the other port caninclude a non-vented cap, such as non-vented cap 180 shown in FIG. 6.The single vented cap can provide the venting for injecting andextracting biological fluid through the penetrable flexible seal 114.

In an example embodiment, the penetrable flexible seal 114 (e.g., rubberstopper) can be removed so that other materials could be added to thebiological-fluid container 102. For instance, in an example embodiment,a user can pull a rubber stopper out of the cap 104 to expose a hole inwhich the rubber stopper was positioned, and then insert other materials(e.g., tissue or fluid(s)) and/or instruments (e.g., a blender orblending apparatus) into the biological-fluid container 102 through thathole.

In accordance with example embodiments, methods to isolate one or morefractions of a biological fluid are provided. An example method includesinjecting biological fluid 140 into biological-fluid container 102through injection port 106 in cap 104, wherein the injection port 106 isconfigured to attach to vented cap 110. The method also includesincubating the biological fluid 140 injected into the biological-fluidcontainer 102 and centrifuging the biological-fluid container 102 toform two or more fractions 142, 144 of the biological fluid. Further,the method includes removing, via a needle 134, one or more fractions142 of the biological fluid from the biological-fluid container 102through the penetrable flexible seal 114 in the cap 104, wherein duringsaid removing the vented cap 110 is attached to the injection port 106and releases pressure from the biological-fluid container 102.

Another example method includes injecting blood 140 intobiological-fluid container 102 through injection port 106 in cap 104.The injection port 106 is configured to attach to vented cap 110, andthe biological-fluid container 102 has a plurality of beads 132 toactivate a protein production in the blood 140. The method furtherincludes incubating the biological-fluid container 102 for a sufficienttime (e.g., about 6, 8, 12, 24, 36, 48 hours or more) and at asufficient temperature (e.g. about 30, 32, 35, 37, 39, 40, or 45° C.) toproduce a therapeutically active protein in the blood. The method alsoincludes centrifuging the biological-fluid container 102 to form a serum142 fraction containing the therapeutically active protein. The methodalso includes removing, via a needle 134, the serum fraction 142 fromthe biological-fluid container 102 through penetrable flexible seal 114while leaving behind other fractions in the biological-fluid container102. During said removing, the first vented cap 110 is attached to theinjection port 106 and releases pressure from the biological-fluidcontainer 102. In an example embodiment, the first vented cap 110 isattached to the injection port 106 during said injecting, and saidinjecting comprises injecting the biological fluid or blood 140 throughthe first vented cap 110 via needle 134.

In accordance with example embodiments, methods for covering abiological-fluid container of a cellular-separation system are provided.An example method includes providing a cap 104 including (i) aninjection port 106 configured for injection of biological fluid throughthe injection port, (ii) an extraction port 108 configured forextraction of biological fluid through the extraction port, (iii) afirst vented cap 110 configured to attach to the injection port 106,(iv) a second vented cap 112 configured to attach to the extraction port108, and (v) a penetrable flexible seal 114 configured for insertion andextraction of biological fluid through the flexible seal. The methodthen includes attaching the cap 104 to a biological-fluid container 102of the cellular-separation system 100.

In accordance with example embodiments, methods for inserting andextracting a biological fluid from a biological-fluid container of acellular-separation system are provided. An example method includesinserting a biological fluid into a biological-fluid container 102through a penetrable flexible seal 114 via a needle 134 or extracting aportion of a biological fluid through the penetrable flexible seal 114via the needle 134. The method further includes venting thecellular-separation system 100 via at least one vented cap (e.g., firstvented cap 110 and/or second vented cap 112) during the insertion orextraction of the biological fluid through the penetrable flexible seal114 via the needle 134.

It should be understood that these methods are intended as examples andother processes and methods to isolate one or more fractions of abiological fluid, for covering a biological fluid of acellular-separation system, and for inserting and extracting abiological fluid from a biological-fluid container of acellular-separation system are disclosed herein. Alternativeimplementations are included within the scope of the examples of thepresent disclosure in which functions can be executed out of order fromthat discussed, including substantially concurrent or in reverse order,depending on the functionality involved, as would be understood by thosereasonably skilled in the art.

The disclosed methods and systems described herein beneficially provideimproved methods and systems for cellular separation. The disclosedmethods and systems provide an improved cap for a biological-fluidcontainer of a cellular-separation system. This improved capbeneficially allows for maintaining sterile conditions inside of abiological-fluid container during the entire process of delivery,incubation, centrifugation, and extraction of biological fluid from thebiological-fluid container. Furthermore, this improved cap beneficiallyallows for an enhanced ability to localize particular points within thebiological-fluid container for extraction. In particular, the penetrableflexible seal of the disclosed cap allows for a large range of motion ofa needle inserted into the biological-fluid container. The range ofmotion is increased compared to existing systems, and beneficiallyallows for an improved ability to localize particular points within thebiological-fluid container for extraction. The improved cap also allowsfor numerous insertion and extraction methods that can be tailored basedon the particular application and/or component of biological fluid to beextracted.

By the term “substantially” it is meant that the recited characteristicneed not be achieved exactly, but that deviations or variations,including for example, tolerances, measurement error, measurementaccuracy limitations and other factors known to skill in the art, canoccur in amounts that do not preclude the effect the characteristic wasintended to provide.

Although the disclosed systems and methods are described primarily withreference to a system that isolates IL-1Ra production in blood, itshould be understood that disclosed systems and methods can be used inconjunction with other biological fluids and for extraction of othercomponents. For instance, the disclosed systems and methods can beimplemented in systems configured to isolate any desired component(s) ofany suitable biological fluid(s).

The term “biological fluid” refers to, including but not limited to,serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicularfluid, seminal fluid, amniotic fluid, milk, whole blood, fractionatedblood, plasma rich platelets, bone marrow, urine, cerebro-spinal fluid,saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissueculture medium, as well as tissue extracts such as homogenized tissue,tumor tissue, adipose tissue, and cellular extracts.

The description of the different advantageous arrangements has beenpresented for purposes of illustration and description, and is notintended to be exhaustive or limited to the embodiments in the formdisclosed. Many modifications and variations will be apparent to thoseof ordinary skill in the art. Furthermore, different advantageousembodiments may describe different advantages as compared to otheradvantageous embodiments. The embodiment or embodiments selected arechosen and described in order to explain the principles of theembodiments, the practical application, and to enable others of ordinaryskill in the art to understand the disclosure for various embodimentswith various modifications as are suited to the particular usecontemplated. As used herein, the term “about” in association with anumerical value means that the value varies up or down by 5%. Forexample, for a value of about 100, means 95 to 105 (or any value between95 and 105).

We claim:
 1. A cap for a biological-fluid container, the cap comprising:an injection port configured for injection of biological fluid throughthe injection port; an extraction port configured for extraction ofbiological fluid through the extraction port; an injection-port capconfigured to attach to the injection port; an extraction-port capconfigured to attach to the extraction port, wherein at least one of theinjection-port cap or the extraction-port cap is a vented cap; and apenetrable flexible seal configured for insertion and extraction ofbiological fluid through the flexible seal, wherein the flexible seal ispresent on the top of the cap for a biological-fluid container.
 2. Thecap of claim 1, wherein the at least one vented cap is configured torelease pressure from the biological-fluid container during insertionand extraction of biological fluid through the flexible seal via aneedle.
 3. The cap of claim 1, wherein the injection port and extractionport each include a cannula configured to extend into thebiological-fluid container.
 4. The cap of claim 1, wherein the injectionport comprises a connection fitting configured to attach directly to asyringe or the injection-port cap, and wherein the extraction portcomprises a connection fitting configured to attach directly to asyringe or the extraction-port cap.
 5. The cap of claim 1, wherein thepenetrable flexible seal comprises a rubber stopper.
 6. The cap of claim1, wherein the injection-port cap and the extraction-port cap are ventedLuer caps.
 7. The cap of claim 1, further comprising a base configuredto attach to the biological-fluid container.
 8. The cap of claim 7,wherein the base is attached to the biological-fluid container, andwherein the cap for a biological-fluid container provides a hermeticseal for the biological-fluid container.
 9. The cap of claim 1, whereinthe injection-port cap is tethered to the injection port, and whereinthe extraction-port cap is tethered to the extraction port.
 10. The capof claim 1, wherein the injection port comprises threading toaccommodate the injection-port cap, and wherein the extraction portcomprises threading to accommodate the extraction-port cap.
 11. The capfor a biological-fluid container of claim 1, wherein the penetrableflexible seal is on the same plane on the cap for a biological-fluidcontainer as the injection port and the extraction port.
 12. Acellular-separation system comprising: a biological-fluid container; anda cap attached to the biological-fluid container, wherein the capcomprises: an injection port configured for injection of biologicalfluid through the injection port; an extraction port configured forextraction of biological fluid through the extraction port; aninjection-port cap configured to attach to the injection port; anextraction-port cap configured to attach to the extraction port, whereinat least one of the injection-port cap or the extraction-port cap is avented cap; and a penetrable flexible seal configured for insertion andextraction of biological fluid through the flexible seal, wherein theflexible seal is present on the top of the cap for a biological-fluidcontainer.
 13. The cellular-separation system of claim 12, wherein thebiological-fluid container comprises a tubular body.
 14. Thecellular-separation system of claim 12, further comprising a pluralityof beads in the biological-fluid container.
 15. The cellular-separationsystem of claim 14, wherein the plurality of beads are suitable toactivate Interleukin-I-Receptor Antagonist Protein.
 16. Thecellular-separation system of claim 12, wherein the at least one ventedcap is configured to release pressure from the biological-fluidcontainer during insertion and extraction of biological fluid throughthe flexible seal via a needle.
 17. The cellular-separation system ofclaim 12, wherein the cap for a biological-fluid container hermeticallyseals the biological-fluid container.
 18. The cellular-separation systemof claim 12, wherein the injection port and extraction port each includea cannula configured to extend into the biological-fluid container. 19.The cellular-separation system of claim 12, wherein the penetrableflexible seal comprises a rubber stopper.
 20. The cellular-separationsystem of claim 12, wherein the injection-port cap and theextraction-port cap are both vented caps.
 21. A method to isolate one ormore fractions of a biological fluid, the method comprising: injecting abiological fluid into a biological-fluid container through an injectionport in a cap of the cellular-separation system of claim 12, wherein theinjection port is configured to attach to a vented cap; incubating thebiological fluid injected into the biological-fluid container;centrifuging the biological-fluid container to form two or morefractions of the biological fluid; and removing one or more fractions ofthe biological fluid from the biological-fluid container through thepenetrable flexible seal in the cap, wherein during said removing thevented cap is attached to the injection port and releases pressure fromthe biological-fluid container.
 22. The method of claim 21, wherein thevented cap is attached to the injection port during said injecting, andwherein said injecting comprises injecting blood through the vented cap.23. The method of claim 21, wherein the biological fluid is blood,wherein the biological-fluid container has a plurality of beads toactivate protein production in the blood, wherein incubating thebiological-fluid container comprises incubating the biological-fluidcontainer for a sufficient time and at a sufficient temperature toproduce a therapeutically active protein in the blood, and whereincentrifuging the biological-fluid container to form two or morefractions of the biological fluid comprises centrifuging thebiological-fluid container to form a serum containing thetherapeutically active protein.